In 1994 this project was initiated based on our ability to functionally transfer the entire 6- deoxyerythronolide B synthase (DEBS) pathway into Streptomyces coelicolor, a genetics friendly (and now genomics-friendly) heterologous host. During the past 5 years our efforts have primarily been focused along two directions. On one hand we have sought to develop genetic and biochemical tools to study and manipulate DEBS with the aim of generating new "unnatural" natural products. At the same time, we have conducted a variety of exploratory studies aimed at uncovering novel properties of modular polyketide synthases (PKSs). Over the next 5 years, we propose to continue our twin-track approach of dissecting, at increasing level of detail, the properties of modular PKSs, and developing useful technology for combinatorial biosynthesis. Our plans include (i) the development and application of genetic screens for DEBS mutants with novel properties; (ii) quantification of the incoming acyl chain and the extender unit specificity of individual modules; (iii) elucidating the precise structural features that influence this specificity; (iv) evaluating the feasibility of introducing new chemistry such as methyltransfer into modules; (v) understanding the mechanistic principles underlying vectorial chain transfer between adjacent modules; (vi) elucidating how macrocycle formation is catalyzed, and the molecular recognition features associated with this amazing reaction; and (vii) (if possible) obtaining high-resolution X-ray crystal structures of DEBS and its variants. In the process we expect to refine established strategies for manipulating the intramodular and intermodular chemistry of modular PKSs, and also develop new ones. These next generation methods are likely to expand the molecular diversity attainable by interfacing genetic engineering with chemistry, and also increase the efficiency/yield with which these complex chemicals are made. Given the re-emerging interest in natural product based drug discovery, our long-term goal is to make polyketide natural products increasingly accessible to chemists and biologists alike.